Anxiety disorders during pregnancy are not routinely assessed in Sri Lanka despite being common and being associated with adverse pregnancy outcomes. Screening can facilitate early detection and management of anxiety and improve pregnancy outcomes. Our aim was to determine the validity of the Sinhala translation of the Perinatal Anxiety Screening Scale (PASS) to detect anxiety among Sri Lankan pregnant women.n = 221) were sequentially recruited and assessed by a psychiatrist until 81 women with anxiety disorder were diagnosed using the International Classification of Diseases-10 criteria (gold standard). The Sinhala translation of the PASS (PASS-S) was administered to all recruited women, including 140 women without anxiety. Receiver-Operating- Characteristic (ROC) analysis was performed, the optimal cut-off score for PASS-S was determined, and its validity was assessed using sensitivity, specificity, predictive values and positive and negative likelihood ratios. Internal consistency was assessed using Cronbach’s alpha. Test-retest and inter-rater reliability for PASS-S score and anxiety classification were assessed using intra class correlation coefficient (ICC) and Cohen’s kappa (k), respectively.A cross-sectional study was conducted in antenatal clinics of a teaching hospital in Colombo District. The PASS was translated to Sinhala using the standard translation/ back-translation method. Pregnant women (The mean age (±SD) of women was 30(±5.8) years, and 53.7% were multiparous. A psychiatrist diagnosed anxiety disorder was made in 37.0% of women, while the PASS-S, at its optimal cut-off of ≥20, classified 37.5% of women as having anxiety disorders. The area under the ROC curve for the PASS-S was 0.96 (95%CI 0.94–0.99). Sensitivity, specificity and positive and negative predictive values of the PASS-S were 0.93 (95% CI 0.84–0.97), 0.90 (95% CI 0.83–0.94), 0.85 (95% CI 0.75–0.90) and 0.95 (95% CI 0.89–0.98), respectively. Positive and negative likelihood ratios were 8.8 (95% CI 5.3–14.5) and 0.08 (95%CI 0.04–0.18), respectively, and the internal consistency was high . Four-factor structures obtained by exploratory factor analysis were “acute anxiety and adjustment”, “social anxiety, specific fears and trauma”, “perfectionism and control” and “general anxiety”.Test-retest reliability was high for the PASS-S score (ICC 0.85[95% CI 0.65–0.96]) and anxiety classification (k 0.77[95% CI 0.34–1.2]). Inter-interviewer reliability was also high (ICC 0.92[95% CI 0.81–0.97] for the PASS-S score and (k0.86 [95% CI 0.59–1.1] for anxiety classification).The Sinhala translation of the PASS is a valid and reliable instrument to screen for anxiety disorders among antenatal women in Sri Lanka. Anxiety symptoms are common during pregnancy and the postpartum period , which cAnxiety during pregnancy in low- and middle-income countries  is as hiLimited evidence suggests that antenatal anxiety is associated with adverse foetal and maternal outcomes including still birth, intrauterine growth restriction (IUGR), prolonged labour, caesarean deliveries, preterm birth, low birth-weight, and low Apgar scores at birth , 12–15. The Perinatal Anxiety Screening Scale (PASS) is an acceptable, psychometrically-sound questionnaire that has performed well in screening for antenatal and postnatal anxiety disorders  includinIn Sri Lanka, the prevalence of adverse pregnancy outcomes remains high despite other maternal health indices being satisfactory. This could be due to causes that are not assessed routinely during pregnancy, including anxiety disorders. However, no valid screening tool is available in Sri Lanka to detect antenatal anxiety disorders. Although the Edinburgh Postnatal Depression Scale (EPDS) which screens for postnatal depression has been used , this inThis was a descriptive cross-sectional study conducted among antenatal women attending hospital antenatal clinics (ANCs). The study was conducted in the ANCs of Colombo South Teaching Hospital (CSTH), which is the second largest tertiary-care institution in Colombo District with a bed strength of 1096 and an annual turnover of 150,000 in-ward patients and 75,000 out-patients. Antenatal women from its catchment area, which is urban/semi-urban, and those referred to it from elsewhere due to risk conditions are registered at ANCs run by three obstetrics units, and they are followed up until the delivery , 29.The study population was pregnant women who attend ANCs of CSTH.Pregnant women attending ANCs at CSTH were recruited to the study irrespective of the trimester of pregnancy . The inclusion criterion was age of at least 18 years. Those with disabilities such as hearing difficulty, visual and speaking problems were excluded. Although diagnosed but currently untreated mental disorders were also an exclusion criterion, no one was excluded based on this criterion.As per the expected sensitivity (70%) and specificity (30%) of the PASS , the calEligible pregnant women were recruited using systematic random sampling with a sampling interval of 5. The sampling frame was the list of registered women in the ANC attendance registry.Participants were given written information detailing the purpose of the study, voluntary participation, procedures and time required for the procedures, potential benefits, discomforts, confidentiality and the ability to terminate participation at participant’s discretion without having to face any adverse consequences. The information was also read out by the Principal Investigator (PI), and queries raised by the participants were clarified. The participants were given an additional 1 h on the average to ask any further questions. The decision to participate in the study was purely voluntary and written informed consent was taken from all participants. The participants were also given contact details of the research team to enable them to clarify any concerns even after the study participation was over.The PI, who is a graduate nurse, checked the eligibility for the study based on the selection criteria by directly questioning the participants and checking the antenatal records. Recruited women were assessed by a psychiatrist as described below. Recruitment continued until 81 women with anxiety disorder were diagnosed.The PASS is a 31-item validated instrument used to screen for anxiety disorders and cover all domains of anxiety in antenatal and postpartum women . Each itThe International Classification of Diseases and Related Health Problems (ICD-10) provides clinical descriptions and diagnostic guidelines of diseases. It also provides the inclusion and exclusion criteria for diagnoses, allowing a degree of flexibility to be retained for diagnostic decisions in clinical setting . DiagnosThe present study was conducted in three phases. Firstly, the PASS was translated into Sinhala and its content, conceptual and semantic validity were assessed. Secondly, the construct validity of the Sinhala translation was examined. Thirdly, the criterion validity and reliability of the Sinhala translation was determined.Translation and adaptation was carried out according to the World Health Organization (WHO) guidelines on the process of translation and adaptation of instruments . Stages Firstly, the English version of PASS was reviewed by a local expert panel to determine its comprehensiveness and appropriateness for local use. This panel consisted of three psychiatrists, an obstetrician, three community physicians and two clinical psychologists. All 31 items of PASS were marked as suitable for screening anxiety disorders in antenatal women by this expert panel. Secondly, two independent bi-lingual experts translated the PASS into Sinhala. The two Sinhala versions were compared by an expert panel, which included a psychiatrist and three psychologists, and the inadequate terminologies/conceptions of the translation and any discrepancies between two Sinhala translation versions were resolved.This first draft of the Sinhala version was back-translated into English by two other independent bi-lingual translators who were blinded to the English PASS. The back-translation was reviewed by an expert panel, which included a psychiatrist and three psychologists, and 28 items were rated as the correct translation, and the other three items, as needing minor revisions. Discrepancies were discussed and resolved. This panel provided consensual approval on the preservation of original English meaning of all items of PASS.The standard translation-back translation method that was followed in the study was similar to how the translation of PASS was done in Bangladesh  and in TThis Sinhala version was pretested among 20 women 18 years of age or older, attending ANCs of another major tertiary care hospital in Colombo District, which is similar in setting and patients to the CSTH, to assess comprehensibility of the questions. Every fifth woman, as per the clinic registration number, was recruited until 20 women were recruited. They were interviewed by the PI using the probing method in cognitive interviewing approach to confirm the clarity of the questionnaire, understanding of the language used and consistency of the answers given.Upon pre-test, the final Sinhala translation of PASS (PASS-S) was checked by an expert panel comprising three psychiatrists, who were not involved in the initial translation process, for its content, conceptual and semantic validity. The final expert panel confirmed all 31 items of the PASS-S to be of appropriate standard to screen for anxiety symptoms of antenatal women in Sri Lanka, of which 30 items were rated as culturally relevant, and one item, as moderately relevant.The PASS-S was piloted among 20 women 18 years of age or older, attending primary-care ANCs at community level, to identify any practical constraints in administering this in such clinics where any antenatal screening is likely to take place, including the convenience of administration, participants’ problems when responding and any other issues. Those who participated in the pre-test and the pilot study were not included in the actual validation study. Although the sample selected for the pilot test was from different ANCs, they were similar in their characteristics to the sample used for the validation study. All procedures conduced in the main study were piloted except for the clinical interview conducted by the psychiatrist. No practical constraints were encountered and the feasibility of implementation of the validation study was confirmed.The construct validity of PASS-S was assessed using the methods described under the section on statistical analysis presented below.Validation of PASS-S against the gold standard was conducted among pregnant women attending ANCs at CSTH.Recruited women were first assessed by a psychiatrist using ICD-10 criteria. A total of 221 women were recruited including 81 with anxiety disorders. This was immediately followed by administration of the PASS-S as an interviewer administered questionnaire by two trained nurses. The time taken to complete an interview using the PASS-S was 6–8 min on average.The findings of the psychiatrist and the nurses were blinded to each other. The PASS-S was re-administered to a randomly-selected 20 women 2–3 days after the initial assessment by the same nurse who administered the PASS-S at the first interview. To another randomly-selected 20 women, the PASS-S was re-administered 2–3 days after the initial assessment by a nurse different from the one who administered the first interview.Descriptive data on participants are reported as numbers and percentages or means and standard deviations (SDs). To examine the construct validity of PASS-S, sample adequacy and suitability for factor analysis were conducted using Kaiser-Meyer-Olkin (KMO) test and Bartlett’s test, respectively. Principal component analysis (PCA) for factor extraction in exploratory factor analysis (EFA) and oblique rotation method which was suggested for correlated variables  were useConfirmatory factor analysis (CFA) was also performed after ensuring that the required assumptions were fulfilled. LISREL 10.2 software was used for this. Additivity test was employed to check whether the scale was an additive scale. A four-factor model was evaluated. The indices used to determine the model fit included the absolute fit indices, relative fit indices and parsimony fit indices. Absolute fit indices were chi-squared test, root mean squared error of approximation (RMSEA), goodness of fit index (GFI), adjusted goodness of fit index (AGFI) and standardized root mean squared residual (SRMR). Relative fit indices were comparative fit index (CFI) and non-normed fit index (NNFI), while parsimony fit indices were parsimony goodness of fit index (PGFI) and parsimonious normed fit index (PNFI).RMSEA values below 0.05 indicate a good fit to data, values between 0.05 and 0.08, an acceptable fit, values between 0.08 and 0.10, a marginal fit and values above 0.10, a poor fit , 34. ForUsing the psychiatrist’s diagnosis as the gold standard, the receiver-operating-characteristic (ROC) curve was graphed for the total scores for PASS-S obtained by participants. The optimal cut-off score of the PASS-S for detecting anxiety in pregnancy was determined using Youden’s index . PerformInternal consistency of the PASS-S was measured using Cronbach’s alpha. Test-retest reliability and inter-rater reliability of the total PASS-S scores were assessed using intra-class correlation coefficient (ICC) for the total PASS-S score, and those of anxiety classification (symptomatic of anxiety disorder/ not symptomatic) were assessed using Cohen’s kappa (k).The mean age ± SD of the recruited antenatal women was 30 ± 5.8 years. Most (74.2%) of the participants had completed primary education and 56.1% were multiparous (Table n = 81) as having anxiety disorder using ICD-10 Criteria. Most women with anxiety had generalized anxiety disorder or phobias (Table The psychiatrist diagnosed 37.0% of women (as Table .Table 2The area under the ROC curve for PASS-S scores was 0.96 (95% CI: 0.94–0.99) Fig. . The optn = 3) had severe anxiety symptoms, while 96.3% (n = 79) had mild to moderate anxiety symptoms. Cronbach’s alpha for internal consistency was 0.95 (95% CI: 0.93–0.96). The ICC for total PASS-S score was 0.85 (95% CI: 0.65–0.96) for test-retest reliability and 0.92 (95% CI: 0.81–0.97) for inter-rater reliability. The Cohen’s kappa for anxiety classification (symptomatic of anxiety disorder/ not symptomatic) was 0.77 (95% CI: 0.34–1.2) for test-retest reliability and 0.86 (95% CI: 0.59–1.1) for inter-rater reliability.According to PASS-S scores, 3.7% of the pregnant women .The KMO test value for sampling adequacy was 0.95. Bartlett’s test of sphericity was significant (χn = 221). An examination of the factor loadings after rotation  explains all the factors included in PASS-S.2 value of 705.57 . RMSEA was 0.056 while SRMR, GFI and AGFI were 0.046, 0.83 and 0.803 respectively. CFI, NNFI, PGFI and PNFI were 0.92, 0.919, 0.716 and 0.766 respectively. The four-factor model was closer to the good fit indices in each absolute, relative, and parsimony fit indices.Fit indices obtained in the CFA indicated that the data fit the hypothesized measurement model perfectly. The four-factor model gave a χThe Sinhala translation of the PASS has produced a valid and a reliable tool to assess anxiety disorder during pregnancy in Sri Lanka. The PASS-S showed high sensitivity, specificity, and predictive values in a local clinic setting. Its test-retest and inter-rater reliability measures were also high. It also showed good content, conceptual and semantic validity similar to those in the two studies conducted in Turkey  and Bangp < 0.00), and was also similar to that of the English PASS. Although the number of factors identified was 4 in the English PASS, the item distribution for the factors slightly varied from the English PASS and PASS-TR. In the English PASS, factor 1 (acute anxiety and adjustment) had items that addressed symptoms of panic disorder, dissociative disorder and adjustment difficulties, factor 2  covered symptoms of general anxiety disorder and phobia, factor 3  included symptoms of obsessive compulsive disorder and post-traumatic stress disorder, and factor 4  had questions to determine social anxiety. However, in the PASS-S, factor direction and the titles of factors were altered. Factors 1–4 were reclassified as “acute anxiety and adjustment” covering symptoms of panic disorders, acute stress and adjustment disorders, “social anxiety, specific fears and trauma” covering symptoms of social anxiety disorder, phobias and post-traumatic stress disorder; “perfectionism and control” covering symptoms of obsessive compulsive disorder and “general anxiety” covering symptoms of generalized anxiety disorder, respectively. These changes were essential components of intercultural adaptation, and the factor structure of the current study reflects specific classification of anxiety disorder in the Sri Lankan context. The factor analysis showed that generalized and acute anxiety symptoms are more prominent (factors loaded under factor 1 of the factor structure) among antenatal women in Sri Lanka while excessive worry and specific fears were prominent anxieties among participants of the English PASS validation study [Sampling adequacy for EFA was excellent (KMO = 0.95) and was similar to the English PASS validation study (KMO = 0.96) . Inter-ion study .The optimal cut-off point of ≥20 for PASS-S was lower than the score of 26 for the English PASS  but highr = 0.74 [r = 0.83 [The internal consistency of the PASS-S was similar to the Turkish PASS , the Banr = 0.74 ) and ther = 0.83 ). Our stThe PASS-S was administered as an interviewer-administered questionnaire to maximize the response rate and to ensure that literacy level of the participants did not influence the responses , 40. TheThe main strength of our study is that the PASS-S was validated against the gold standard of a psychiatric diagnosis of anxiety disorders. Ours is only the second study to do this, the first being validation of the PASS-TR against both Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV) criteria and ICD-10 criteria . FurtherThe good validity and reliability of the PASS-S make it a suitable tool to be used in clinical practice and in research, and may even be used routinely to screen for anxiety disorders among antenatal women in Sri Lanka. Further studies to assess the feasibility of using the PASS-S as a screening tool to detect anxiety disorders during routine antenatal care are recommended. Validation of the PASS in Tamil, the other local language in Sri Lanka, is also recommended.The PASS-S is a valid and reliable instrument to assess anxiety during pregnancy among Sinhala speaking women, and may be useful in routine assessment of anxiety in such women.Additional file 1: Table S1. Result of content/consensual validity-Appropriateness of factor structure use in Sri Lankan context. Table S2. Comparison of Factor Structure in PASS and PASS-S. Table S3. Fit indices, their description and cut-off values used for interpreting model fit in CFA (1).


The Oral Health Impact Profile-14 (OHIP-14) was designed to assess patients’ perception of the impact of oral disorders on their quality of life (QoL). Although the OHIP-14 is now frequently used in patients with head and neck cancer, data related to its measurement properties in this population are scarce. The aim of the present study was to evaluate the reliability and validity of the OHIP-14 in a sample of Serbian patients with head and neck cancer. Data were available for 345 patients , with head and neck cancer. All patients completed the OHIP-14 and the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-H&N35. Factor analyses, internal consistency reliability , and construct validity were analyzed.p< 0.01). Patients with a tumor stage 0-II also had lower OHIP-14 scores compared to those who had a tumor stage III-IV (p< 0.01). The OHIP-14 correlated significantly with the QLQ-C30 and QLQ-H&N35 scales. The factor analyses confirmed that 14 OHIP items were measuring a single underlying factor. Cronbach’s α coefficient was 0.98 and corrected item-total correlations ranged 0.77-0.93. Lower OHIP-14 scores  were more frequently present among patients who had only surgery as a therapeutic procedure compared to those who had surgery accompanied with radio- and chemotherapy ( As a unidimensional instrument, the OHIP-14 provides oral QoL assessments with sound internal consistency reliability and construct validity among patients with head and neck cancer. Key words:Quality of life, oral health, psychometrics, reliability, validity. Over the past 30 years, patient reported outcome (PRO) measures have become standard methods for providing additional information on how patients perceive the impacts of their health condition and its treatment on everyday living . EspeciaHead and neck cancer (HNC) affect different locations of the head and neck , with phet al.  is designed to assess individuals’ perception of the impact of oral disorders on their QoL . This is- ParticipantsThis is a cross-sectional, psychometric study that included patients treated for HNC, who were admitted to Department of ENT with maxillofacial surgery of Clinical Hospital Centre Zemun, Belgrade, Serbia. As a sample of convenience, all patients were recruited during pre-scheduled assessments when visiting a doctor for follow-up care. The participation was on a voluntary basis and the only exclusion criterion was inability to read and/or write. To all patients was first explained the aim of the study and all included patients provided written informed consent before participating in the study. The instruments (see below) were self-completed by included patients.Complete data for analyses were available for 345 patients with HNC , aged 30-92 years .- InstrumentsOHIP-14: The OHIP-14 has 14 items with answers rated on a 5-point Likert scale (from 1 = never to 5 = very often) to indicate a level of different problems related to oral health over in the last 12 months . The totEuropean Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-H&N35: The QLQ-C30 is a 30-item, cancer-specific instrument for QoL assessments . It has Scores for all scales of the both instruments are calculated by linear transformation of raw scores into a 0–100 score, where scores of 100 represent the best outcomes on the QLQ-C30 functioning scales and the worst outcomes on the QLQ-C30 and QLQ-H&N35 scales. The Serbian versions were provided by the EORTC Group.- Statistical analysisStatistical analyses included the assessment of descriptive characteristics, factor structure , internal consistency reliability, and construct validity . To carry out the analyses, SPSS Statistics Version 24 and AMOS Version 18 were used.Factor structure: To explore the factor structure , the whole sample was randomly split. Principal axis factoring (PAF) with Promax (oblique) extraction method, as an EFA method, was used with the data of 162 participants . Afterwards, 14 items were subjected to a maximum likelihood solution with the second sample of 183 participants  was employed to test CFA. A series mean repletion was used to handle missing data as the amount was negligible per item (0.3–0.9%). Absolute model fit to the data for the CFA was evaluated using the comparative fit index (CFI), Tucker-Lewis index (TLI), and root mean square error of approximation (RMSEA) with the following cut-offs : TLI andDescriptives and internal consistency reliability: The distribution of mean scores, standard deviation (SD), and percentage floor and ceiling effects were calculated. A floor and ceiling effect were defined as the percentage of individuals with the best and worst results respectfully. More than 15% of participants with the highest or lowest score on one particular scale were considered as a relevant effect. Internal consistency reliability was evaluated using Cronbach’s α coefficient and the values of α ≥ 0.7 were considered accepTable.Know-group validity: A t-test and one-way analysis of variance (ANOVA) were used to test for differences in OHIP-14 scores among different groups for which it would be expected that OHIP-14 scores differ. It was hypothesized that patients with cancer located other-then-oral regions would have lower scores  then those with oral cavity and pharyngeal cancer (epi/oro/hypo pharynx) or laryngeal cancer as well as those patients who had only surgery as a therapeutic procedure compared to those who had surgery accompanied with radio- and chemotherapy patients, and patients with a TNM stage 0-II compared to a TNM stage III-IV.Convergent/discriminant validity: Convergent and discriminant validity was evaluated with Pearson correlation coefficients between the OHIP-14 and the QLQ-C30 and QLQ-H&N35 scales. Correlation coefficients ranging 0.1–0.3 were considered low, those 0.31–0.5 moderate, and those exceeding 0.5 high.- Factor Structurep < 0.01). The PAF analysis revealed a single underlying factor explaining 79.7% of the total variance, with all 14 items having high factor loadings (ranging 0.66-0.95). Afterwards, a CFA analysis of model showed the following fit indexes (χ2 (df) = 285.49 [77]; TLI = 0.93, CFI = 0.94, and RMSEA = 0.12, with all standardized regression weights being statistically significant . On the same sample was also tested the model of seven correlated factors  and Bartlett’s Test of Sphericity (χ2 (df) = 3286.54 [91],  factors ,16 with  . In addition, patients with a TNM stage 0-II had significantly lower OHIP-14 scores compared to those who had a TNM stage III-IV (t (df) = 11.05 [343]; p < 0.01). Based on ANOVA, statistically significant differences in the OHIP-14 score were found when comparing patients with three different localizations of cancer .Patients who had only surgery as a therapeutic procedure had significantly lower OHIP-14 scores compared to those patients who had surgery accompanied with radio- and chemotherapy (t (df) = -15.99 [343]; - Convergent/Discriminant ValidityExcept with the Teeth (r = 0.29), Diarrhea (r = 0.22), and Weight gain (r = -0.27) scale, the OHIP-14 correlated moderately to highly with the other QLQ-C30 and QLQ-H&N35 scales .The OHIP-14 was developed as short instrument to represent functional limitation, physical pain, psychological discomfort, physical, psychological and social disability, and overall handicap in relation to oral disorders . AvailabFurther analyses showed that the OHIP-14 has appropriate aspects of construct validity, namely known-group and convergent/discriminant validity, what is also in line with the previous findings from studies with other populations ,23. BaseThere are some limitations to be acknowledged regarding the methodology of our study. It should be noted that only patients who agreed to participate were included and the sampling was convenient, thus those who may have less or more advanced cancer stages might not have participated. Additional psychometric aspects, such as screening properties, test-retest, responsiveness or predictive validity, and diagnostic value were also not tested.Summarizing, the OHIP-14 is measuring a single underlying factor of oral health among patients with HNC, with sound internal consistency and construct validity. This study generalizes its use and opens the space for further use of OHIP-14 with these patients in research and clinical setting.


Cordyceps militaris, has gained much attention due to its various clinical functions. Previous reports of L-alanine addition could significantly improve cordycepin production, but the molecular mechanism remains unknown. In this study, transcriptome analysis of C. militaris with doubled cordycepin production induced by L-alanine addition provides an insight into the flexibility of the cordycepin network. The biopathways of energy generation and amino acid conversion were activated so that cordycepin substrate generation was consequently improved. Specific genes of rate-limiting enzymes in these pathways, as well as related transcription factors, were figured out. Two key Zn2Cys6-type transcription factors CmTf1 and CmTf2 were verified to play the roles of doubling the cordycepin production by overexpression of their coding genes in C. militaris wild type. These results provide a complete map of the cordycepin network in C. militaris with a distinct understanding of the flexibility of joints, giving a better foundation for increasing cordycepin yield and strain breeding in the future.Cordycepin, isolated from the traditional medicinal fungus  Cordyceps militaris has been popular in East Asia for centuries as a kind of edible and medicinal fungus. Recently, the application of various bioactive metabolites isolated from C. militaris led to rapid development in food, pharmaceutical, and cosmetics industries  were not only involved in the development of C. militaris fruiting  medium. After fermentation at 25°C with 150 rpm for 5 days, the medium was supplemented with 8 g/L L-alanine and continued to shake for 3 days before the harvest.Since the static fermentation of After fermentation, the mycelia were lyophilized and ground to powder. Two grams of dry powder was weighted from each sample to perform HPLC detection. HPLC assay was performed on Model LC2000 Liquid Chromatography System  equipped with an ULTIMATE AQ-C18 HPLC Column . The analysis conditions were as follows: mobile phase, 85% ultra-pure water and 15% methanol (v/v); flow rate, 1 ml/min; detection wavelength, 260 nm; injection volume, 20 μl. A standard cordycepin curve was generated using 0.02–0.25 μg/ml cordycepin standard . The cordycepin yield was calculated using the detected peak area according to the standard curve. The cordycepin concentration of mycelia presented in the study was calculated by normalizing in the equal biomass.Total RNA was extracted from 100 mg of frozen mycelium pellets using the E.Z.N.A. Fungal RNA Miniprep Kit . After DNase I treatment, the quality of RNA was assessed by a NanoDrop and Agilent 2100. Oligo(dT) was used to isolate mRNA. The mRNAs were fragmented and used as templates to synthesize cDNA. Short fragments were purified and processed by end repair, single-nucleotide adenine addition, and adapter connection. Suitable fragments were selected for PCR amplification. The quantification and qualification of the sample library were performed by an Agilent 2100 Bioanalyzer  and an ABI StepOnePlus Real-Time PCR System , and further analyzed by following the operating instruction. The library was sequenced using the Illumina HiSeq by BGI .C. militaris CM01 (NCBI accession number. AEVU00000000) using HISAT  , adaptor-polluted reads, and reads with a high content of unknown bases (unknown bases are more than 5%) using BGI  in-house software with the quality system phred64 were removed and clean reads were stored in FASTQ  format. -X 1000) . StringT-X 1000)  (version-X 1000)  (version-X 1000)  (version-X 1000)  (version-X 1000)   was based on the Poisson distribution method (PoissonDis)  were detected using edgeR criteria in R  accordinssonDis) , and thessonDis)  and KEGGssonDis) .tef1) gene (NW_006271969.1) as an internal control for each sample . All primers used for the quantitative real-time PCR (qRT-PCR) are listed in h sample , the relT method .NW_006271970.1) and CCM_01481 (accession number NW_006271969.1) gene were cloned from C. militaris CM10 by primers Tf1F/Tf1R and Tf2F/Tf2R  and constructed into plasmid p390-blpR-sgRNA-cmcas9-gfp  to build p390-blpR-CmTf1/2. Then the overexpressed vectors were transformed into Agrobacterium tumefaciens AGL-1  by electroporation. The A. tumefaciens-mediated transformation (ATMT) was used to build the CmTf1/2 overexpression C. militaris as described in a previous study (The transcription factors CCM_02568 gene (accession number f2F/Tf2R . These gcas9-gfp  by XbaI/us study .y = 1e-08x+0.001 (R2 = 0.9994), the cordycepin extraction yield detected by HPLC was obtained. The total amount of 36.9 and 41.0 mg/g of cordycepin was extracted from the dry mycelia harvested from the CMsI group, while the other group was 18.0 and 19.3 mg/g , suggesting its importance in the metabolism of supplementing L-alanine.GO classification and functional enrichment were performed for the gene annotation. A total of 523, 338, and 279 DEGs were assigned to the ontologies “biological process,” “molecular function,” and “cellular component,” respectively . Over 10P-values of 12 pathways were less than 0.05 (P < 0.01).To have a better understanding of the interactions of functional genes by pathway-based analysis, all the genes were mapped to the KEGG database to categorize the biological functions of the DEGs. Specific gene enrichment was observed in 118 pathways. Twenty-one pathways were showing DEG enrichment (965 genes), and the cluster “Global and overview maps” was the largest group . Clusters for “Amino acid metabolism” , “Carbohydrate metabolism” , “Transport and catabolism” , “Signal transduction” , and “Translation”  were followed . Moreovehan 0.05 . The patDEGs that took part in the pathway of autophagy regulation (KEGG pathway ko04140) were not shown enrichment, indicating that large-scale cell starvation did not occur in the fermentation. It also indicated that the increased yield of cordycepin was caused by the interaction affection of L-alanine in the metabolic network. A network map of substance interactions affected by L-alanine addition was obtained by the comparison between the transcriptome data and the KEGG database . InteracAs shown in the network, L-cysteine and L-glutamate played key roles in substance transformation after adding L-alanine. Other amino acids, such as glutamine, glycine, and L-aspartate, which played an important role in this network had been reported to be involved in the purine pathway . It indi1 to detect putative transcription factors. Output candidates were further verified by InterPro2. A total of 94 putative TFs from 983 DEGs (10.5%) was predicted and classified into different TF families. These TF families contained Zn2Cys6 1  Zn2Cys6 , HMG  and CCM_01481 . A previous work reported  that theC. militaris was predicted  and CCM_08873 , annotated as plasma membrane H+-ATPase, were significantly up-regulated, which matched those of the previous report. CCM_06062, the significantly up-regulated putative DEG in C. militaris, was annotated as pyruvate kinase (classified as EC 2.7.1.40) according to sequence homology analysis. Its highly up-regulated transcriptional level will lead to the function of improving ATP generation. Since adenine could be obtained by the over-dephosphorylation of ATP, CCM_06062 indirectly increased the production of adenine, which is a plausible candidate substance of cordycepin. Other than adenine, AMP was also a plausible candidate substrate of cordycepin because previous report showed that adenylate kinase was a key enzyme in cordycepin synthesis , while only one DEG, CCM_07799 , was significantly up-regulated in the transcriptome data. A previous study speculated that the synthesis location for cordycepin and purine is separated . The up-cns1 , and CCM_04437, annotated as cns2 , were both significantly up-regulated at similar levels. However, the transcriptional level of the gene CCM_04438, annotated as cns3 , was only slightly increased. 3′-AMP could be also produced by phosphoesterase (EC 3.1.4.16) from 2′,3′-cAMP, which was generated during mRNA degradation. Though the phosphoesterase gene was not determined in C. militaris, 2′,3′-cAMP has been verified to be involved in cordycepin production  were major facilitator superfamily (MFS) transporters , and 16 To further verify the RNA sequencing data, 13 genes represented four different pathways discussed in this study were selected to perform qRT-PCR validation. The qRT-PCR results  showed tP < 0.01) of the fold change between qRT-PCR and RNA-Seq results.Of these genes, seven genes, which were predicted to participate in cordycepin metabolism as described in C. militaris. The protein Cns3 was also able to synthesize pentostatin, a purine analog that functions as a safeguard of cordycepin to balance its in vivo concentration to avoid the toxic effect from a high intracellular level of cordycepin , CCM_05735 , CCM_06273 , and CCM_07510 ], which were involved in the repair mechanism of the synthesis-dependent strand annealing in the homologous recombination pathway (KEGG ko03440), were significantly up-regulated in RNA sequencing data and the qRT-PCR results. This might indicate that the powerful DNA repair mechanism in C. militaris neutralized the toxicity of cordycepin, which causes DNA breaks.Cordycepin, as the analog of adenosine, is capable of blocking the synthesis of DNA or RNA in inflammatory cells. Nevertheless, a high dose of cordycepin showed no obvious effect on mycelial growth in rdycepin . The traTwo other genes, CCM_06968  and CCM_04351 , which were distinctly different at the transcriptional level and annotated to join in the mitogen-activated protein kinase signaling pathway ko04011, were randomly picked to be verified by qRT-PCR. Their qRT-PCR data were also consistent with the RNA-Seq result.Overall, these qRT-PCR results showed that the DEGs transcription level was consistent with the data obtained by transcriptome analysis.C. militaris genome by ATMT to obtain the CmTf1/2 overexpressed strains and renamed as CM10Tf1/CM10Tf2. The growth rate and pigment accumulation  gene CCM_06768 could transform IMP into adenylosuccinate, and then the adenylosuccinate lyase (EC 4.3.2.2) gene CCM_05789 will perform the reaction of producing AMP. CCM_06768 was significantly up-regulated while CCM_05789 was not, but the transcriptional level of CCM_05789 was higher than that of 90% DEGs. The gene CCM_00088, annotated as APRTase (EC 2.4.2.7) and conducted the reaction from adenine into AMP, also had a high transcriptional level but only showed slightly up-regulated while compared to the control. The high transcriptional level of these genes indicated that they might be involved in cordycepin synthesis, even though they were not significantly up-regulated in the experimental group with L-alanine addition. Previous studies have noted the importance of adenylate kinase  and APRT+-ATPase genes CCM_01006 and CCM_08873 were highly up-regulated to improve the cordycepin production. This discovery accords with the GO classification and TFs results, suggesting that L-alanine activates additional expression of transporters known to affect energy generation and metabolic substance conversion.On the other hand, not only the phenotypic character but also the KEGG classification and ko01100 results indicated that the energy generation was acting an important role in the cordycepin production. From a cellular perspective, pyruvate not only joins in ketone body formation as a precursor but also takes part in ATP generation. In this study, high-level gene expression of pyruvate kinase gene CCM_06062 indicated that it joined in cordycepin metabolism by improving ATP generation. As consistent with the previous report , H+-ATPaAs mentioned above , the dircns1 and cns2 were significantly up-regulated. This result suggested that the dosage of additive L-alanine in this study was enough to sustain improvement of the cordycepin production. Contrary to expectations, this study showed that gene cns3 was not significantly up-regulated between samples, indicating that adding L-alanine could not significantly activate the expression of Cns3. One possible hypothesis is that most of substrate 3′-AMP is synthesized from 2′,3′-cAMP through the undetermined enzyme EC 3.1.4.16 during mRNA degradation.Cordycepin was reported to be synthesized from adenosine by a three-gene cassette . AdenosiC. militaris. In brief, the addition of L-alanine increased the cordycepin yield by activating the pathway of energy generation and amino acid interconversion. In the biopathway of energy generation, the activated pyruvate kinase and plasma membrane H+-ATPase directly increased the accumulation of ATP, while the Zn2Cys6 TFs (CCM_02568 and CCM_01481) promoted activities in some unknown ways to generate more energy. The consumption of extra ATP could generate a high amount of ADP and AMP, which are precursors of adenosine. In the other biopathway, L-alanine joined in the interconversion of amino acids, the high amount of L-alanine accelerates the formation of amino acids. Through the histidine metabolism, these amino acids indirectly promoted the accumulation of IMP, which is also the precursor of adenosine. The enzymes of the reactions between AMP, IMP, and their intermediate products  to adenosine performed an active stage because of the sufficient AMP and IMP. Thus, the production of cordycepin was increased by excess adenosine and energy.Above all, the combination of transcriptome data in this study and the data from previous reports revealed a reliable metabolic network map of cordycepin production affected by L-alanine addition in the fermentation of C. militaris activated by L-alanine addition. Up-regulating the transcriptome level of genes involved in the bio-pathways of energy generation and amino acid conversion was the major reason for the overproduction of cordycepin. Two Zn2Cys6-type transcription factors were verified to be related to the rate-limiting steps by increasing the copy number of their coding genes in C. militaris wild type. As the whole cordycepin metabolic network map was drawn, this is one step toward discovering the flexibility of cordycepin network and providing a reference for genomic editing to improve cordycepin production as well as other compounds of medicinal value in C. militaris.We presented the transcriptome analysis of the metabolic network of cordycepin in PRJNA413637.The datasets generated for this study can be found in the NCBI Accession: B-XC performed data analysis and was a major contributor in drafting the work. TW helped revise the manuscript critically for important intellectual content. B-XC and L-NX performed RNA extraction and qRT-PCR. Q-WZ and Z-WY supervised and B-XC in sequencing data analysis. YZ and YY performed cordycepin detection. FY performed strain culturing. L-QG and J-FL agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors approved the last version of the manuscript to be published.FY was employed by Guangzhou Alchemy Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The biological response of multi-walled carbon nanotubes (MWNTs) is related to their physicochemical properties and a thorough MWNT characterization should accompany an assessment of their biological activity, including their potential toxicity. Beyond characterizing the physicochemical properties of MWNTs from different sources or manufacturers, it is also important to characterize different production lots of the same MWNT product from the same vendor . Herein, we present a comprehensive physicochemical characterization of two lots of commercial pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs) used to study the response of mammalian macrophages to MWNTs. There were many similarities between the physicochemical properties of the two lots of cMWNTs and neither significantly diminished the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 μg cMWNTs/mL). Conversely, several physicochemical properties of the two lots of pMWNTs were different; notably, the newer lot of pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot. Furthermore, a 72-h half maximal inhibitory concentration (IC-50) of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of pMWNTs. These results demonstrate that subtle physicochemical differences can lead to significantly dissimilar cellular responses, and that production-lot consistency must be considered when assessing the toxicity of MWNTs. The unique physicochemical properties of multi-walled carbon nanotubes (MWNTs) lend themselves to a variety of industrial and biomedical applications  processes are the dominant modes of high-volume production because of low energy consumption, low waste generation, and the ability to tailor MWNT properties such as their outer diameters . Even soent used . Unfortuent used ,32,33,34ent used ,38,39,40ent used ,31,32,41For these reasons, it is not uncommon for researchers to perform extensive material characterizations of commercially obtained MWNTs and to compare their findings with the manufacturer’s product specifications ,43,44,45Herein, we present a comprehensive physicochemical characterization of two lots of commercial, CVD-synthesized pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs) used to study the biological responses of mammalian macrophages to MWNTs. There were many similarities between the physicochemical properties of the two lots of cMWNT powders and of bovine serum albumin (BSA)-coated cMWNT suspensions prepared with each cMWNT powder. Equally importantly, BSA-cMWNT suspensions prepared with the two cMWNT powders did not significantly diminish the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 μg cMWNTs/mL). The new production lot of cMWNTs is therefore a strong candidate to be a suitable replacement for the original lot of cMWNTs when it is exhausted. Conversely, several physicochemical properties of the two lots of pMWNT powders and of the BSA-coated pMWNT suspensions prepared with each powder were different. The new pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot of pMWNTs. Also, the concentration of pMWNTs that could be suspended by BSA with the new lot of pMWNTs was significantly lower relative to the original lot of pMWNTs. Most importantly, while the 24-h proliferation of RAW 264.7 macrophages cultured with the original BSA-pMWNT suspensions were statistically similar to the proliferation of cells observed with the two BSA-cMWNT suspensions, the 24-h proliferation of RAW 264.7 cells incubated with BSA-pMWNT suspensions prepared with the new pMWNTs was not. Specifically, the 24-h proliferation of cells incubated with BSA-suspensions of the new pMWNTs at 100 µg/mL was ~20% lower relative to BSA-suspensions of the original pMWNTs at 100 µg/mL, even though the amount of the new pMWNTs accumulated by cells was ~16% less relative to the amount of original pMWNTs accumulated by cells. Furthermore, a 72-h half maximal inhibitory concentration (IC-50) of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of BSA-pMWNTs, making the 2018-pMWNTs significantly more toxic than the original pMWNT lot. These results demonstrate that subtle physicochemical differences can have a significant effect on the response of biological cells to a MWNT product, and that production-lot consistency must be considered when assessing the toxicity or biomedical performance of MWNTs.® Integral water purification system . Phosphate buffered saline  was sterilized by autoclaving at 121 °C for 45 min. Stock solutions of 100 mg/mL BSA were prepared by dissolving 10 g of BSA in 100 mL of deionized water and adjusting the pH to 7.4. Working solutions of 0.10 mg/mL BSA were prepared by diluting stock BSA solutions with aqueous 10 mM HEPES and filtering the solutions through a 0.22-μm pore membrane; stock and working solutions of BSA were stored at 4 °C in the dark.CVD-synthesized pMWNTs and cMWNTs were purchased directly from the manufacturer. The original pMWNT and cMWNT powders were acquired in 2015 and a second purchase of the exact same products was obtained in 2018; herein, they are denoted as 2015-pMWNTs, 2015-cMWNTs, 2018-pMWNTs, and 2018-cMWNTs. All MWNTs were stored at room temperature in the dark. Caution, a fine-particulate respirator and other appropriate personal protective equipment should be worn when handling dry MWNT powders. Dulbecco’s modified Eagle medium (DMEM) was purchased from Gibco , fetal bovine serum (FBS) was purchased from Atlanta Biologicals , and BSA, penicillin , streptomycin (10 mg/mL), and all other chemicals were purchased from Millipore Sigma ; all chemicals were used as-received. Deionized water (18.3 MΩ-cm) was obtained using a Milli-QThe elemental content of each MWNT powder was determined according to a previously described combustion analysis technique with the exception that all samples were vacuum dried for 4 h at 100 °C prior to analysis . The CHNThe sonication and centrifugation protocol described in our previous works ,51 was u® standards  and ten consecutive 30-s runs were taken per measurement at 25 °C. The hydrodynamic diameter was calculated using a viscosity and refractive index of 0.8872 cP and 1.330, respectively for deionized water, and an absorption and refractive index of 0.010 and 1.891, respectively, for MWNTs. Zeta potential values were also determined for purified BSA-coated MWNT suspensions that were diluted 1:10 with deionized water. In addition, DLS and zeta potential analyses were performed periodically on purified MWNT suspensions stored at 4 °C. Typically, MWNT suspensions were stable in storage for months, indicated by the lack of aggregates detected by DLS and constant zeta potential results.Dynamic light scattering (DLS) and zeta potential analyses were used as part of a quality control routine for the preparation of all MWNT suspensions, as previously described ,51,54. T® TIB-71™; Manassas, VA, USA). RAW 264.7 cells were grown in DMEM supplemented with 1.5 mg/mL sodium bicarbonate, 10 mM HEPES (pH 7.4), and 10% (v/v) FBS; the standard incubation conditions were 37 °C in a 5% CO2 and 95% air environment.Abelson murine leukemia-virus transformed RAW 264.7 macrophages were purchased from the American Type Culture Collection (ATCCv/v) FBS, 200 units/mL penicillin, and 0.2 mg/mL streptomycin. The result is a test medium with the same concentration of 10 mM HEPES and 10% FBS as the control medium. ~3.5 × 104 RAW 264.7 cells/well were seeded in 48-well plates and incubated at 37 °C overnight before the medium was replaced with freshly prepared control medium or test medium containing MWNTs at a specified concentration, and incubated for 24 or 72 h. At the end of the incubation, cells were washed three times with fresh medium, two times with PBS, air-dried, and fixed with 4% (w/v) paraformaldehyde in PBS. Cell proliferation was determined using a BioTek Synergy Mx plate reader and the standardized crystal violet assay as detailed in our previous work, where it was demonstrated that MWNTs do not interfere with the assay  of surface –O–H groups, –O–H moieties in carboxylic acid groups, water chemisorbed to MWNTs, and/or residual moisture in the KBr pellet  that are polarized by adjacent oxygenated groups  of carbonyls and carboxyl groups [FTIR spectroscopy was employed to gain more specific insight into the surface oxygen species present on the four MWNT products. The FTIR spectra shown in r pellet ,96,97. Td groups ,96,97,98l groups ,97, as wl groups , the FTIvide infra). Moreover, as shown in A set of pMWNTs and cMWNTs with similar dimensions and purities was purchased in 2015 for evaluating the response of functionalized MWNTs to mammalian macrophages. Lot-acceptance testing was performed using a combustion analysis technique to evaluate the carbon purity of the pMWNTs and cMWNTs. As shown in vide infra). Additionally, TEM and HR-TEM imaging did not reveal any significant differences in the inside and outside diameters of the four MWNTs , and doping with elements other than carbon [D/IG ratio.A perfect crystalline carbon nanotube comprises only hexagonal rings of speatments ,99. Defen carbon . Both th3-carbon region and the π–π* region (3 or OH·) of single-walled carbon nanotubes, and observed increases in surface carboxylic acids or esters , but they did not observe any changes in toxicity with human A549 adenocarcinoma-derived alveolar epithelial cells and THP-1 leukemia-derived peripheral blood monocytes [The percentages of carbon and oxygen determined by XPS were consistent with the elemental analysis results obtained using a combustion analysis technique, except for the lower amount of surface oxygen detected by XPS for the 2018-pMWNTs . Analysi* region , whereas* region . FTIR sp* region . XPS andonocytes . Herein,Determining the fundamental origin(s) of a cytotoxic response to a MWNT sample is a complex endeavor because many MWNT physicochemical determinants are interrelated and it is difficult to systematically decouple them ; for exaA more established tenet is that structural defects, surface chemistry, surface curvature, and the surface area of a MWNT are decisive factors involved in the dynamic formation of a protein corona ,109. In TGA and Raman analyses suggest the 2018-pMWNTs had more defects relative to the 2015-pMWNTs , and the2/g) was roughly twice that of the 2015-cMWNTs (~144 m2/g). However, the Raman spectral profiles and the ID/IG ratios of the 2015-cMWNTs and 2018-cMWNTs  were highly comparable  were in close agreement , as weremparable , as weremparable  and XPS mparable . Finallyoxylated .Suspensions of BSA-coated cMWNTs prepared with the 2015-cMWNT and 2018-cMWNT powders were also quite similar. As shown in A comprehensive physicochemical characterization of two commercial lots of CVD-synthesized pMWNTs and cMWNTs revealed many similarities between the two cMWNT products and several key differences between the two pMWNT products. The 2018-pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the 2015-pMWNTs. Additionally, the concentration of pMWNTs that could be suspended by BSA with the 2018-pMWNTs was significantly lower relative to the 2015-pMWNTs. Most importantly, while the 24-h proliferation of RAW 264.7 macrophages cultured with BSA-suspensions of 2015-pMWNTs were statistically similar to the proliferation of cells observed with the two BSA-cMWNT suspensions, the 24-h proliferation of RAW 264.7 cells incubated with BSA-suspensions of 2018-pMWNTs was not. Specifically, the 24-h proliferation of cells incubated with BSA-suspensions of 2018-pMWNTs at 100 µg/mL was ~20% lower relative to BSA-suspensions of 2015-pMWNTs at 100 µg/mL, even though the amount of the 2018-pMWNTs accumulated by cells was ~16% less relative to the amount of 2015-pMWNTs accumulated by cells. Furthermore, a 72-h IC-50 of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with BSA-suspensions of 2018-pMWNTs, making the 2018-pMWNTs significantly more toxic than the 2015-pMWNTs.The differences in the surface chemistry and structural defects of the 2018-pMWNTs relative to the 2015-pMWNTs likely influenced the protein corona that was formed when BSA-pMWNT suspensions were prepared, which in turn could affect the binding and subsequent accumulation of the 2018-pMWNTs by RAW 264.7 cells. Reactive structural defects, a key determinant of toxicity, also likely influenced the diminished 24-h proliferation of RAW 264.7 cells, as well as, the 72-h toxicity observed with the 2018-pMWNTs. This work therefore demonstrates (i) the difficulty in assessing the role of a single physicochemical property of a MWNT product to an observed biological response, (ii) that subtle physicochemical differences can have a significant effect on the response of biological cells to a MWNT product, and (iii) that production-lot consistency must be considered when assessing the toxicity or biological activity of MWNTs and other carbon nanomaterials.


Lactobacillus have gained increasing interest in counteracting Candida infections for their proven anti-adhesive properties and safety profile. In the present study, liposomes (LP-BS) or liposomes coated with hyaluronic acid (HY-LP-BS) were prepared in the presence of the BS isolated from the vaginal strain Lactobacillus crispatus BC1 and characterized in terms of size, ζ potential, stability and mucoadhesion. The anti-biofilm activity of free BS, LP-BS and HY-LP-BS was investigated against different Candida albicans and non-albicans strains , clinically isolated from patients affected by VVC. The inhibition of biofilm formation and the dispersal of pre-formed biofilm were evaluated. The obtained phospholipid vesicles showed suitable size for vaginal application and good stability over the storage period. HY-LP-BS exhibited good mucoadhesive properties and the best anti-biofilm profile, both in preventing or limiting the surface colonization by a broad spectrum of Candida species. In conclusion, the formulation of a novel antifungal agent derived from the vaginal microbiota into mucoadhesive nanocarriers appears to be a promising biotherapeutic strategy to counteract vulvovaginal candidiasis.The emergence of resistance to antifungal drugs has made the treatment of vulvovaginal candidiasis (VVC) very challenging. Among natural substances, biosurfactants (BS) produced by  Candida albicans, but non-albicans Candida species, such as C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis, have been also identified as etiological agents. While the epidemiology of infection varies among these species, their general virulence attributes appear mostly conserved. Concerning virulence, a majority of invasive infections are related to the microbial ability to live and grow in a sessile form. Biofilms are closely packed communities of microbial cells that adhere to surfaces, such as tissues and implanted medical devices. Candida species are commonly able to produce biofilms and acquire drug resistance; drug sequestration by the biofilm matrix is responsible for much of the antifungal tolerance. Consequently, infections involving biofilm establishment are challenging to cure [Candida species as a consequence of extensive use of antifungal azoles [Lactobacillus, may offer the opportunity to deliver health benefits, as probiotics are part of healthy microbiota and therefore are beneficial for humans and safe for therapeutic purposes [Lactobacillus crispatus BC1 was found to interfere with the adhesion of various Candida isolates to human cervical epithelial cells; furthermore, BS intravaginal inoculation in a murine experimental model was shown to be safe and effective in reducing leukocyte influx in the case of C. albicans infection [L. crispatus species plays a crucial role in maintaining vaginal eubiosis, exerting beneficial properties that have already been extensively addressed in previous studies [Vulvovaginal candidiasis (VVC) is a multifactorial infectious disease of the lower female reproductive tract and it represents one of the most common vaginal infections worldwide. More than 90% of VVC cases are caused by  to cure ,2. Consil azoles ,4, new tl azoles , have bel azoles . In partpurposes ,8. In a  studies ,12,13,14Candida agents based on vaginal probiotics, the development of suitable carriers could further improve the treatment. In recent years, liposomes have demonstrated their potential to counteract local infections due to their ability to guarantee a modified release of the active substance. Such a property ensures an adequate concentration of the active drug at the administration site [Together with the use of alternative anti-ion site ,16. At tion site , includiion site . Extendeion site  and alsoion site . Moreoveion site . Due to Lactobacillus crispatus BC1, were evaluated for their ability to inhibit biofilm formation and disperse pre-formed biofilm of different C. albicans and non-albicans clinically isolated strains.In the present work, two novel strategies have been merged: the choice of a new potential antifungal agent derived from the human healthy microbiota and the use of mucoadhesive nanocarriers for its vaginal delivery. Two different types of liposomes, prepared in the presence of the BS isolated from the vaginal strain L. crispatus BC1 (BC1-BS) to interfere with the adhesion of Candida spp. to HeLa cells. Pathogen adhesion represents the first step in the mucosal colonization and the subsequent biofilm development. BC1-BS was chemically characterized and found to contain an amino acid portion attributable to tyrosine, serine, proline, glycine and arginine linked to fatty acids, namely β-hydroxytridecanoic acid (3-OH-C13), β-hydroxytetradecanoic acid (3-OH-C14), β-hydroxypentadecanoic acid (3-OH-C15) and β-hydroxyhexadecanoic acid (3-OH-C16). Moreover, BC1-BS is able to reduce the surface tension and it showed a critical micellar concentration equal to 2 mg/mL. BC1-BS revealed a good anti-adhesion activity tested against clinically relevant Candida spp. for concentrations up to 1.25 mg/mL, which was also found not to be cytotoxic to HeLa cells [In a previous work, we demonstrated the ability of BS produced by the vaginal strain La cells . Thus, cp < 0.05), probably as a consequence of polymer adsorption on the lipid bilayer. This result is in agreement with our recent findings [When nanocarriers are intended for local vaginal application, their size and surface charge play an important role in improving the treatment. In the present study, conventional liposomes (LP) or liposomes coated with hyaluronic acid  were prepared; phospholipid vesicles containing BC1-BS were developed, giving rise to LP-BS and HY-LP-BS. The main physicochemical properties of the developed vesicles are summarized in findings  and prevfindings . Specififindings ,23.p < 0.05). This behavior is probably correlated with the particular composition of the biosurfactant. In fact, as reported in De Gregorio et al., BC1-BS was characterized by the presence of different fatty acids and amino acids, including arginine [On the other hand, for both liposomes and hyalurosomes, the presence of BC1-BS (LP-BS and HY-LP-BS) led to the formation of smaller structures with respect to the phospholipid vesicles prepared without biosurfactant   is a measure of the width of unimodal size distributions . In our p > 0.05), probably due to the low polymer concentration (0.01% w/v). This datum is in agreement with that of a previous work, in which similar results were obtained when 0.2% w/v polymer concentration was used [p < 0.05). This result can be attributable to the presence of positively charged residues of arginine on the BS structure that could interact with the anionic phosphate groups of phosphatidylcholine, providing a more positive structure. Our data agree with the findings of Duangjit and co-workers, who observed an increase of positive charges on the liposome surface when it contained a cationic surfactant [Phospholipid vesicles were characterized by negative ζ potential values, as a consequence of the presence of phosphatidylcholine phosphate groups that in saline solution (pH = 6.0) were negatively charged, in agreement with other published works ,22,36,37was used . Finallyrfactant .The effect of storage on the main properties of phospholipid vesicles was assessed by monitoring the changes of size and PDI over a storage period of 180 days at 4–8 °C. The vesicle variation in terms of size is reported in p < 0.05). HY-LP showed a greater interaction with mucin compared to LP ; probably as a consequence of the interaction between the polymer and the mucin chains. As reported in several studies, hyaluronic acid can interact with mucin in virtue of the chain entanglement and physical interlock with mucus and the presence of many hydrophilic groups that can establish hydrogen bonds [p < 0.05), even if no significant difference was observed between liposomes and hyalurosomes (p > 0.05). The improvement of mucoadhesion could be ascribed to the presence of different aminoacids in BS structure able to interact with mucin. Specifically, as described before, the positively charged residues of arginine could interact with the negatively charged sialic acid (pKa = 2.6) and sulfate residues of mucin.A prerequisite for successful topical vaginal therapy is the formulation’s ability to guarantee a prolonged contact with the vaginal mucosa . For thien bonds . MoreoveC. albicans biofilm in vitro has been correlated with in vivo and ex-vivo models [C. albicans  and five non-albicans  strains, clinically isolated from patients affected by VVC. Moreover, two different experiments, namely the inhibition of biofilm formation and the dispersal of pre-formed biofilm, were taken into account.Since the formation of o models , in the Candida suspensions were simultaneously incubated with free BC1-BS, LP-BS and HY-LP-BS and the biofilms were allowed to develop for 72 h, which is the average time required for biofilm maturation [Candida strains with inhibition rates ranging from 44% to 66% for C. albicans strains  showed no activity. This result is in agreement with a previous study, which demonstrated the capability of liposomes to favor the anti-biofilm activity against Staphylococcus aureus of BS isolated from another Lactobacillus vaginal strain [The presence of BS in conventional liposomes (LP-BS) significantly enhanced their ability to interfere with the biofilm formation of seven l strain . These fl strain .p < 0.05) the biofilm formation of three C. albicans strains (inhibition rate of ~30%) and four non-albicans strains (inhibition rate of 15–28%), with the only exception being C. tropicalis SO24. Hyaluronic acid is known to possess antimicrobial activity against Candida [Candida isolate biofilm, probably due to the combined contribution of BS and HY. In particular, HY-LP-BS strongly impaired the formation of biofilms by all tested C. albicans strains, with inhibition rates above 78%. The impact on non-albicans strains was slightly lower, with inhibition percentages ranging from 57% to 85%. These results suggest that the proposed vaginal formulation can be effective in preventing or limiting the surface colonization by a broad spectrum of Candida species, and thus reducing the severity of infection.In order to further ameliorate the anti-biofilm profile, a coating of hyaluronic acid was added to the formulation (HY-LP-BS). Interestingly, in this case the carrier itself (HY-LP) slightly reduced , suggested that the employment of hyaluronic acid can be a valid strategy also to deal with fungal biofilm. Notably, HY-LP-BS revealed higher dispersal performance against all Candida strains tested as compared to free BS (p < 0.05), with eradication rates of 53–72% for C. albicans strains  and sodium hyaluronate  were provided from Lipoid GmbH  and Farmalabor , respectively. Mucin , crystal violet, L-cysteine hydrochloride monohydrate and all solvents were from Sigma-Aldrich .De Man, Rogosa and Sharpe medium (MRS) was supplied by Difco  and Sabouraud dextrose medium (SD) from Oxoid . GasPak EZ was purchased from Becton Dickinson and Company .2HPO4, 0.19 g/L KH2PO4 and 8 g/L NaCl, pH = 7.4.Ultrapure water (18.2 MΏ cm) was obtained by means of a MilliQ apparatus by Millipore . The phosphate buffer (PBS) was prepared with the following composition: 2.38 g/L NaL. crispatus BC1 isolation was done from the vaginal swab of a healthy premenopausal woman and the protocol was approved by the Ethics Committee of the University of Bologna, Italy (52/2014/U/Tess) [w/v of MRS powder and 0.05% w/v L-cysteine; lactobacilli were grown at 37 °C for 24 h in anaerobic jars containing GasPak EZ./U/Tess) . BC1 strCandida spp. were employed in the present study, namely C. albicans SO11-SO4, C. glabrata SO17, C. lusitaniae SO22, C. tropicalis SO24, C. krusei SO26 and C. parapsilosis SO27. They all belong to a collection of yeasts isolated from vaginal swabs of premenopausal, VVC affected women during routine diagnostic procedures at the Microbiology Laboratory of Sant’Orsola-Malpighi University Hospital of Bologna, Italy. Candida isolates were grown in SD medium aerobically at 35 °C for 24–48 h [Nine clinical isolates of  24–48 h .L. crispatus BC1 cell surface as previously reported [g for 15 min. The cell pellet was washed twice in sterile distilled water, resuspended in 240 mL of sterile PBS and gently stirred at room temperature for 2 h to release the cell-bound BS. The sample was then centrifuged and the supernatant filtered through a 0.22 μm pore size filter . The obtained solution was subjected to dialysis against demineralized water in a Cellu-Sep© membrane  for 24 h at room temperature, and freeze-dried at 0.01 atm and −45 °C . About 60 mg of lyophilized BS was obtained from 1 L of L. crispatus BC1 culture. As reported by De Gregorio et al. [L. crispatus BC1 possessed a lipopeptidic structure and a critical micelle concentration of 2 mg/mL, evaluated by Fourier-transform infrared spectroscopy and mass spectrometric analysis and by the ring method using a tensiometer equipped with a platinum ring, respectively.BS was isolated from reported . Brieflyo et al. , BS prod3-CH3OH . The organic phase was evaporated in a round-bottomed flask by using a rotatory evaporator  under reduced pressure (80 mbar) at 55 °C, 210 rpm for 120 min. At the end of this period, a dry lipid film was obtained and hydrated by using the rotatory evaporator  with 10 mL of saline solution (NaCl 0.9% w/v) for 1 h. HY-LP were prepared through rehydration of the lipid film with a sodium hyaluronate solution, obtained by dissolving the polymer in saline solution (0.1 mg/mL) for 30 min under stirring at 200 rpm. For the preparation of LP and HY-LP containing BC1-BS, the biosurfactant was dissolved in the saline solution (1.25 mg/mL) without or with hyaluronic acid. To reduce and homogenize vesicle size, all the suspensions were extruded 10 times through a polycarbonate membrane with a pore size of 200 nm .For the preparation of liposomes (LP) and hyalurosomes (HY-LP), the previously reported film rehydration and extrusion method was employed with some modifications ,54. SpecThe prepared suspensions were named as follows: LP and LP-BS for liposomes, without or with BC1-BS, respectively; HY-LP and HY-LP-BS for hyalurosomes without or with BC1-BS, respectively.v/v) in ultrapure water . Size and PDI were obtained through photon-correlation spectroscopy (PCS) using a Brookhaven 90-PLUS instrument  with He-Ne laser beam at a wavelength of 532 nm (scattering angle of 90°). A Malvern Zetasizer 3000 HS instrument  was used for ζ potential measurement. All the experiments were performed in triplicate.For the determination of phospholipid vesicle size, PDI and ζ potential, suspensions were diluted , aliquots of vesicle suspensions were diluted in ultrapure water as described in w/v) for 6 h. Subsequently, the mucin dispersion was centrifuged at 7500 rpm  for 20 min. The supernatant was isolated, mixed with the different vesicle suspensions (1:1 v/v) and vortexed for 1 min. After 3 h, samples were opportunely diluted in water and the turbidity was immediately measured at 650 nm through UV–Visible Spectrophotometer . The absorbance (ABS) of mucin dispersion itself and vesicle suspension without mucin were also recorded as controls. All the experiments were performed in triplicate.Mucoadhesive properties were investigated by measuring the turbidity of the suspensions containing phospholipid vesicles together with mucin ,39,40,55The anti-biofilm potential of free BS, LP-BS and HY-LP-BS was assessed by considering two different mechanisms of action: the inhibition of biofilm formation and the dispersal of pre-formed biofilm .Candida suspensions were prepared in SD medium at a final concentration of 106 CFU/mL and used as starting inoculum for both assays. BS was solubilized in sterile saline at 1.25 mg/mL as in the liposomal formulations and filtered through a 0.22 μm pore size filter.Candida suspension and 100 μL of BS in the following forms: (i) free BS, (ii) LP-BS and (iii) HY-LP-BS. Plain liposomes (LP) and plain hyalurosomes (HY-LP) were also tested for comparison. Controls contained 100 μL of fungal suspension and 100 μL of PBS. Wells filled with SD only served as blank, while wells containing SD and phospholipid vesicles were included as sterility controls.For the inhibition assay, sterile 96 multi-well flat-bottomed plates  were filled with 100 μL of w/v) in 12% ethanol for 5 min. After washing the wells with PBS for three times to remove the excess stain, the dye bound to adherent yeasts was resolubilized with 200 μL of ethanol and the absorbance was measured at 595 nm (ABS 595) .The multi-well plates were incubated at 35 °C with shaking (100 rpm) for 72 h to allow biofilm development. Afterwards, the liquid culture was removed and biofilm formation was quantified through crystal violet staining. Briefly, adherent cells were washed twice with 200 μL of PBS, fixed with 200 μL of absolute ethanol for 5 min and stained with 2% crystal violet ):Candida suspensions or SD medium only. After 72 h of incubation  the liquid cultures were removed, leaving only adherent biofilm in the wells. Biofilms were then treated with 100 μL of SD and 100 μL of free BS, LP-BS or HY-LP-BS. Control wells were supplied with SD only. LP and HY-LP were tested for comparison; blank and sterility controls were also included. Plates were further incubated at 35 °C with shaking (100 rpm) for 48 h and biofilm quantification was performed through the protocol described above. The biofilm eradication was expressed in percentage relative to the untreated control wells, as reported above (Equation (1)).For the eradication assay, the 96 multi-well plates were inoculated with 200 μL of t-test was applied for the comparison of two means, one-way ANOVA followed by Bonferroni correction was used for multiple comparison. GraphPad Prims version 9.0.0 for Windows  was employed for statistical analyses and differences were deemed significant for p < 0.05.All results were expressed as mean ± standard deviation (SD). Student’s L. crispatus BC1 was indagated as an alternative natural substance to counteract biofilms formed by clinically relevant Candida spp., which are often associated with both drug-resistance and recurrent outcomes. The BS was found to be active towards C. albicans and non-albicans strains, especially in inhibiting the formation of fungal biofilms. In order to favor the vaginal delivery and optimize its biological activity, two different lipidic nanocarriers, liposomes and hyalurosomes, containing the BS were successfully developed. LP-BS and HY-LP-BS revealed optimal sizes to target the vaginal mucosa and good ability to bind mucin, which is an important requisite to assure prolonged permanence of a locally delivered formulation in the site of action. Moreover, they were stable over the storage period of 180 days. Notably, the inclusion of BS inside liposomal formulations allowed enhancing of its anti-biofilm activity. In particular, HY-LP-BS revealed the best profile both in inhibiting Candida biofilms’ formation and in dispersing pre-formed biofilms. Although other studies are required to deeply investigate the potential employment of such formulations in humans, the evaluation of anti-biofilm properties represents a first step in the research and development of new biotherapeutic approaches to counteract VVC. Our preliminary results underline that BS-containing liposomes can be a promising strategy to contain vulvovaginal Candida infections.In the present work a biosurfactant from the vaginal strain


MiR-486 knockout mice have a strong tendency of radiation-induced carcinogenesis. In conclusion, miR-486 inhibits the proliferation of lymphoma cells and tumorigenesis induced by radiation through targeting IGF2BP3.Ionizing radiation is one of the common environmental carcinogens. miRNAs play critical roles in the processes of tumor occurrence, development, metastasis. However, the relationship between radiation-induced carcinogenesis and miRNA rarely reported. This study is aimed to investigate the effect of miRNAs on radiation-induced carcinogenesis. In this study we established the radiation-induced thymic lymphoma mice model. By using miRNA array of RTL tissue and predicting for miRNAs target genes, a miRNA-mRNA crosstalk network was established. Based on this network, we identified a critical miRNA, miR-486, which was the most down-regulated in the radiation-induced carcinogenesis. Then the function of miR-486 was confirmed by using knockout mice and cellular experiments. As a result, miR-486 could inhibit proliferation of mouse lymphoma cells by targeting IGF2BP3 mRNA. The adenovirus over-expression miR-486 vector reduced tumorigenesis  Carcinogenesis could be induced by environmental factors which mainly include physical, chemical, and biological factors, and ionizing radiation is one of the common physical carcinogens –3. RadiaIn recent years, the miRNA is followed with powerful interests. miRNAs play critical roles in the processes of tumor occurrence, development, metastasis, and miRNA generation , 9. HoweWith the development in molecular biology and bioinformatics, more and more studies have verified the roles of miRNAs in cancer. The identification of miRNA-mRNA regulatory modules has proven to be important for understanding cellular systems. It is already found that one miRNA could target multiple mRNAs meanwhile one mRNA could be regulated by several miRNAs . TherefoIn this study, we performed genome-wide mRNA and miRNA expression profiling studies between RTL and normal thymus tissues. By constructing regulatory network between miRNAs and their targets, we screened out the miR-486 as the potential regulator in the process of RTL. Further, the effect of miR-486 on mouse lymphoma cells was investigated and its target gene was identified.60Co γ-ray whole body irradiation were described as our previous work  and non-irradiation group (n = 100) were examined daily. Subsequently, RTL tissues and parallel non-irradiated thymus tissues were randomly selected as paired samples. RNAs and proteins were extracted to quantify their expression.Four-week-old female BALB/c mice , China) were housed in a specific pathogen free facility. The RTL model and ous work . In shorOnce establishment of RTL, the mice were sacrificed after being anaesthetized with 1% pentobarbital sodium. Then the tissues of RTL and normal thymus were removed to divide into three portions. One portion was placed in 10% formalin, and subsequently was performed H&E staining and immunohistochemical staining. Another part of samples was extracted RNA and proteins to detect their expression by RT-PCR and Western blotting. The remains were conducted Affymetrix miRNA chip and mRNA sequencing.2 with 1640 Medium (PAA Laboratories) containing 10% fetal bovine serum (PAA Laboratories). MiR-486 mimics, miR-NC mimics (miR-negative control mimics), miR-486 ASO (miR-486 antisense oligo), and miR-NC ASO (miR-negative control antisense oligo) were obtained from Gene pharma company . The cells were planked originally in the 24 wells plate with density of 0.5–2 × 105/well. When the planked cells reach to 80%, miRNAs were transfected using Lipofectamine 2000 (Invitrogen). Specific procedure of transfection was in accordance with previous study (Mouse lymphoma cells line (EL4), obtained from Chinese Academy of Sciences , was cultured at 37°C in a humidified atmosphere of 5% COus study .The cell viability was detected by CCK-8 assay. Firstly, EL4 cells were seeded in a 96-well plate with the density of 5,000/well. Twenty-four hours after transfection the plates were added CCK-8 reagent . Culturing 1 h, then the OD value was measured to indicate cell viability.The apoptosis of EL4 cells were determined by flow cytometry. Twenty-four hours after irradiation of 8 Gy, the EL4 cells were digested and stained using Annexin V-FITC/PI Cell Apoptosis Detection Kit  for 20 min, then flow cytometry was used to analyze apoptosis rate of EL4 cells.Total RNA was isolated from thymic lymphoma tissues and normal thymus tissues using Trizol  according to the manufacturer’s instructions . Reversehttp://microrna.sanger.ac.uk/sequences/, AgilentTechnolo gies) Chip (8×60K). The chip contains probes for human miRNA  and human virus miRNA , which basically covers the currently known human miRNA. This chip is highly sensitive and specific, and can also distinguish mature and precursor miRNAs.Normal and RITL tissues were sent to Capital Bío Company  for miRNA biochip analysis. The miRNA gene chip used in this experiment is a commercial miRNA oligonucleotide expression profile designed by Agilent based on the miRNA sequence of the database  p-value ≤0.05 and Fold Change ≥5 in up-regulated mRNAs; (2) p-value ≤0.05 and Fold Change ≤0.2 in down-regulated mRNAs.http://gencomp.bio.unipd.it/magia/start), a Pearson correlation analysis was conducted. calculation for the parameter setting (P < 0.05). Then we can select the miRNA/target stored in the TargetSCan prediction library Gene association database, and imported the txt file. MAGIA software will integrate the miRNA and mRNA differential expression profile chip data to obtain mutually regulated miRNA-mRNA set. The node status and degree distribution of the miRNA-mRNA regulatory network could be analyzed. The distribution of a node refers to the number of adjacent nodes or connected edges that the node catch. The more neighbors associated with a node means that the influence on other factors in the network is more extensive.The MAGIA online software was used to conduct correlation research on the miRNA-mRNA expression. First, the standardized data of the differential miRNA and mRNA is converted into.txt format. In this study, the same sample is used for the detection of miRNA and mRNA chip. Using the MAGIA official website (The murine IGF2BP3 over-expression vector was generated by introducing the full length murine IGF2BP3 cDNA into the pcDNA3.1 vector (Invitrogen) in a method like many published papers , 15. AllLuciferase system was used to detect activity of miR-486 in IGF2BP3 3’ UTR. First, the cells were transfected the suitable plasmid in the 24-well plate. Then, cells were collected to test luciferase assay 48 h after transfection with a reporter plasmid containing IGF2BP3 wt-3’UTR and a plasmid expressing renilla luciferase. Using the luciferase detection kit  luciferase detection was performed based on the manufacturer’s protocol .IGF2BP3 secreted from EL4 cells was detected by ELISA. Briefly, the supernate of EL4 cells was collected to measure the contents of IGF2BP3 by ELISA kit . According to the establishment of standard curve, secreted IGF2BP3 level could be quantified.The fixed femur and spleen were thoroughly washed using 0.01 M PBS (pH 7.4) and embedded in paraffin. Paraffin sections cut at a 4 μm thickness for HE. The changes of histopathology were visualized by optical microscope (×200). Immunohistochemistry analyses were performed using paraffin sections. IGF2BP3 expression was investigated. The tissues were incubated in primary antibodies overnight at 4°C and then in horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive cells were visualized using DAB.To gain insights into the biological functions of these miRNA target genes, the Gene Ontology (GO) classification was performed. Kyoto Encyclopedia of Genes Genomes (KEGG) was used to analyze the potential pathway of miRNA target genes. The online based software GENECODIS was utilized for those functional annotation .6) were subcutaneously injected into the backs of NOD/SCID mice. The tumor formation incidences and the tumor size were measured twice weekly for 30 days.48 hours after infection with the ad-virus or negative control, EL4 cells . As a result, 63 miRNAs were screened out, of which 44 miRNAs were up-regulated and 17 miRNAs were down-regulated  and a total of 1,070 target genes were obtained. Combined the predicted genes and achieved mRNA expression profile, the genuine miRNA targets were finally revealed. As a result, 761 miRNA target gene pairs with an inverse correlation of expression were formed and miRNA-mRNA regulatory network was accordingly established . Then, 1 × 1060Co γ-ray whole body irradiation were described in our previous work. Compared with the wild-type mice, the tumor incidence of miR-486 knockout mice were significantly increased, indicating that miR-486 knockout promoted the occurrence of radiation carcinogenesis , we test the biological function of miR-486 and found that overexpression of miR-486 could significantly inhibit the proliferation of EL4 cells, confirming that miR-486 is a critical cancer suppressor. We investigated the target mRNA of miR-486 by using miRNA-mRNA correlation analysis, and found that 37 associated mRNAs were involved, in which IGF2BP3 was recognized as the most significant gene. Luciferase experiments further demonstrated that the suppressive effect of miR-486 was dependent on IGF2BP3 mRNA 3’UTR activity, verifying that miR-486 targets IGF2BP3 mRNA. Considering that miR-486 contained two complementary sequences combining with IGF2BP3 mRNA 3 ‘UTR, we investigated which one is the functional area of miR-486. With construction of different areas of the mutant IGF2BP3 mRNA 3’UTR region, we demonstrated that both two complementary sequences (896-903bp and 928-934bp) play an inhibitory role in IGF2BP3 expression.in vivo. These results suggested that miR-486 exhibited a strong suppressive effect on lymphomagenesis in vivo. In addition, miR-486 knockout mice were established, and RIL model was produced as described. Compared with the wild-type mice, the tumor incidence of miR-486 knockout mice were significantly increased, indicating that miR-486 knockout promoted the occurrence of radiation carcinogenesis. The tumor volumes of miR-486 knockout mice were larger than those of wild-type mice, which indicated that miR-486 knockout could promote tumor growth in vivo. The immunohistochemistry results showed that the positive rate of Ki67 in tumor tissues of miR-486 knockout mice was significantly higher than that of the control group, indicating that miR-486 knockout could promote tumor proliferation. And the positive rates of IGF2BP3 and IGF2 were also higher than that of wild type mice, indicating that by miR-486 knockout, the inhibitory effect on IGF2BP3 was significantly attenuated.To better understand the function of miR-486, we established adenovirus over-expression miR-486 vector and found that the EL4/Ad- miR-486 cells formed smaller tumors than these in the EL4/Ad-NC group. Moreover, the number of IGF2BP3+ and IGF2+ cells was decreased in miR-486 group compared with the NC group Above experiments and results had demonstrated that microRNA-miRNA network was indeed a novel and effective strategy to find important target miRNA in various of disease including radiation-induced tumorigenesis. Moreover, the role of miR-486 was also confirmed in the development of RTL. Actually, in recent years, many studies have also shown that miR-486 plays a very important role in the occurrence and development of a variety of tumors. For example, Chen H found that the deletion of the miR-486 genome promotes the progression of gastric cancer, and the expression level of miR-486 has a certain reference value for the prognosis of patients with gastric adenocarcinoma . In lungThe original contributions presented in the study are included in the article/All animal experiments conformed to the National Institute of Health Guide for the Care and Use of Laboratory Animals’ , with the approval of the Laboratory Animal Center of the Second Military Medical University, Shanghai. The approval ID for this study was SMMU-20187321. Written informed consent was obtained from the owners for the participation of their animals in this study.CL, FG, and HL designed the study. HZ, SD, PX, JD, and TL performed the experiments. YY and YC analyzed the data. HZ and HL wrote the paper. JC supported the fund assistance. All authors contributed to the article and approved the submitted version.This study was supported in part by the grants from the National Natural Science Foundation of China (No. 81872559 and No. 11635014) and Shanghai Sailing Program (No. 18YF1429200), Natural Science Foundation of Shanghai (No.18ZR1449700).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.